Negative selection and stringency modulation in phage-assisted continuous evolution

Abstract

Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to substantially accelerate laboratory evolution experiments. In this work, we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule-controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counterselection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ∼10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates while maintaining wild type-like levels of activity.

Figure 1. Overview of PACE with real-time monitoring

During PACE, host E. coli cells flow continuously into a fixed-volume vessel (a “lagoon”) containing a population of filamentous bacteriophages that encode a library of evolving proteins. The lagoon is continuously drained to a waste container after passing through an in-line luminescence monitor that measures expression from a geneIII-luciferase cassette on the AP. Dilution occurs faster than cell division but slower than phage replication. Each phage carries a protein-encoding gene to be evolved instead of a phage gene (gene III) that is required for infection. Phage encoding active library members trigger expression of gene III on the AP in proportion to the desired activity and consequently produce infectious progeny. Phage encoding less active library members produce fewer infectious progeny and are lost by dilution.

Figure 2. Drift cassette enables ATc-dependent, activity-independent phage propagation

(a-c) Cells harboring APs with the indicated gene III-luxAB expression cassettes served as recipients for phage propagation experiments using SP-T7_WT. Data show representative single measurements of phage concentrations (n = 1). (a) Using P_tet to induce gene III expression with ATc prior to phage infection inhibits infection and results in minimal phage propagation and low phage titers. (b) Using P_psp to express gene III only after infection takes place results in robust activity-independent phage propagation and high phage titers. (c) Infection- and ATc-dependent gene III expression using P_psp-tet enables robust, activity-independent propagation. (d) Recipient cells carrying a drift plasmid (DP) and a P_T7-gene III AP were used to propagate a mixture of SP-T7_WT (wild-type T7 RNAP, high activity) and SP-T7_Dead (D812G mutant T7 RNAP, no activity) at a ratio of 1:10 (“Mix” in (e)). (e) In the absence of ATc, SP-T7_WT (WT) is rapidly enriched over the inactive D812G mutant polymerase (D) and a rapid increase in luciferase signal is observed. (f) At an intermediate ATc concentration (150 ng/mL), SP-T7_WT is enriched at a slower rate, concurrent with a slower rise in luciferase signal. (g) At the highest ATc concentration (400 ng/mL), SP-T7_WT is not enriched and baseline luciferase signal is observed. After ending ATc supplementation at t = 8 h, SP-T7_{WT is} rapidly enriched and the luciferase signal rapidly rises.

Figure 3. Dominant negative pIII-neg is a potent inhibitor of phage propagation

(a) Recipient cells carrying a P_T7-gene III AP and a P_T3-gene III-neg AP_neg in which the theophylline riboswitch controls gene III-neg expression were used to propagate a 1:10 mixture (“mix” in (b)) of SP-T7_Spec (specific for P_T7, “spec”) and SP-T7_Prom (promiscuous on both P_T7 and P_T3, “prom”), respectively. (b) At a high theophylline concentration (1000 μM), the promiscuous T7 RNAP SP is rapidly depleted and the specific T7 RNAP SP quickly takes over the lagoon, concomitant with a sharp rise in luciferase signal from P_T7. (c) At an intermediate theophylline concentration, the promiscuous T7 RNAP SP slowly washes out and is gradually replaced by the specific T7 RNAP SP. (d) In the absence of theophylline, the promiscuous T7 RNAP SP propagates unhindered and the lagoon maintains the starting ratio of the inoculated phage. Upon addition of high concentrations of theophylline to this lagoon at t = 12 h, a rapid washout of the promiscuous T7 RNAP SP takes place, with a rebound in luciferase signal consistent with specific T7 RNAP SP enrichment. In (b)-(d), data show representative single measurements (n = 1).

Figure 4. Continuous evolution of T3-specific RNAP variants

PACE of T7 RNAP variants that recognize P_T3 and reject P_T7 was performed in three contiguous stages of differing stringency (top). The P_T3 and P_T7 bars conceptually represent amounts of gene III (red) or gene III-neg (blue) expressed for a given amount of polymerase activity. At t = 0, host cells were E. coli containing the DP and a P_T3-gene III AP. For the first 12 hours, 200 ng/mL ATc was added to the lagoon to reduce selection stringency to zero, enabling drift. At t = 12 h, the concentration of ATc was reduced to 20 ng/mL to increase the stringency of positive selection. At t = 28 h, host cells were switched to E. coli harboring an MP, a P_T3-gene III AP and a riboswitch-controlled P_T7-gene III-neg AP_neg. At t = 32 h, 1 mM theophylline was added to increase negative selection stringency. At t = 52 h, host cells were switched to E. coli containing an MP, a reduced RBS P_T3-gene III AP, and an enhanced RBS/increased origin copy number P_T7-gene III-neg AP_neg to further increase negative and positive selection stringency. The in-line luminescence monitor was used throughout to infer population fitness (center). Gene expression activities on the T7 and T3 promoters of randomly chosen clones from each stage were measured (bottom). Gene expression data show mean values ± s.e. for two replicates. See Supplementary Figure 4 for mutations present in each clone. N = no RNAP; T7 = wild-type T7 RNAP.

Figure 5. Analysis of evolved T7 RNAP mutations that confer P_T3 specificity

(a) The gene expression activity in cells of T7 RNAP variants containing subsets of mutations found in clones described in Fig. 5 are shown on the T7 promoter (blue bars) and the T3 promoter (red bars). Gene expression data represent mean values ± s.e. for two replicates. (b) Location of evolved mutations. The T7 promoter DNA is rendered as dark blue and light purple surfaces, with light purple denoting nucleotide differences between the T7 and T3 promoters. Cyan spheres identify evolved mutations that enable P_T3 recognition. Red spheres identify mutations that evolved during negative selection that contribute to specific recognition of P_T3 over P_T7. Magenta spheres represent a conserved cluster of mutually exclusive mutations evolved in clones following negative selection. The sequences of the T7 and T3 promoters are shown at the bottom, with the differences in red.